Acute rheumatic fever (ARF) is the major cause of heart disease in children around the world. The disease is rampant in developing countries where prevalence rates of rheumatic heart disease may be as high as 35-40 per thousand individuals. By one estimate, it affects nearly six millon school-age children in India. Although the incidence of ARF in the United States and other Western countries declined markedly during the later half of the twentieth century, there has been a recent remarkable resurgence of the disease in the United States. Hence, the need for a safe and effective vaccine is urgent and serious.
Streptococci are a group of bacteria with the capacity to grow in chains. Many varieties are part of the normal bacterial flora in humans and are not especially harmful. However, a particular subgroup of streptococcal bacteria, called group A and represented by Streptococcus pyogenes, is a human pathogen. Between 20 and 30 millon cases of group A streptococcal infections occur every year in the United States alone. These cases include infections of the skin and throat, forms of pneumonia and a more recently identified disease resembling toxic shock. The most common infection is acute streptococcal pharyngitis, or strep throat, which occurs predominantly in school-age children. Strep throat qualifies as a major worldwide health problem if judged only by time lost from school and work and by the amount spent on related doctor's fees.
Strep throat's toll is much greater, however. In as many as 4% of the pharyngitis cases that are untreated or treated ineffectively, the strep infection leads to ARF. Current attempts to revent ARF rely on treatment of the pharyngitis with antibiotics. During a recent outbreak of ARF in Utah, only a fourth of the patients sought health care prior to the onset of symptoms, and only a third recalled a recent sore throat. The finding that ARF may follow a subclinical infection in such a high percentage of individuals and the fact that access to health care in developing countries is not widely available serve to underscore the need for a safe and effective vaccine against group A streptococci.
The causal relationship between streptococcal pharyngitis and ARF was established over 50 years ago, yet the mechanism of the pathogenesis of the disease remains unclear. It is widely held that ARF is an autoimmune disease, and that in the susceptible host the infection triggers an immune response that leads to inflammatory and sometimes destructive changes in target tissues. Streptococci have been shown to contain antigens that are immunologically cross-reactive with host tissues and heart-cross-reactive antibodies from patients with rheumatic fever have been shown to react with streptococci. However, it was also shown that sera from patients with uncomplicated pharyngitis also may contain heart-cross-reactive antibodies, yet these patients do not develop clinical evidence of carditis. Until the significance of tissue-cross-reactive antibodies in the pathogenesis of ARF is better understood, there remains a need to exclude potentially harmful epitopes from vaccine preparations.
The surface M protein of group A streptococci is the major virulence factor and protective antigen of these organisms, group A streptococci have developed a system for avoiding some of the antimicrobial defenses of a human host. Strains of streptococci that are rich in M protein evade phagocytosis by PMNs and multiply in non-immune blood. Yet, resistance to an infection by these bacteria is possible if the host's body can produce opsonic antibodies directed against the M protein. Such antibodies will neutralize the protective capacity of the M protein and allow the streptococcus to be engulfed and destroyed by phagocytes. The development of secretory or mucosal antibodies as opposed to serum opsonic antibodies, are also now suspected of playing an important role in preventing streptococcal infections.
A major obstacle to effective vaccine development has been the very large number of M protein serotypes. See, Stollerman, "Rheumatic Fever and Streptococcal Infection, Grune & Stratton (1975). These are reported to number about 82 to date and more can be expected to be identified.
It has been shown that antibodies against one serotype do not necessarily offer protection against others although some do cross-react with others. Immunity then appears to be type or sero-specific and optimal vaccines would require that most of the serotypes be represented. The concept of "rheumatogenic" and "non-rheumatogenic" organisms is supported by multiple surveillance studies over many years and in diverse areas of the world. Thus, there are probably about 12-15 serotypes responsible for most cases of ARF. Some of these are types 1, 3, 5, 6, 14, 18, 19, 24, 27 and 29.
To assist in a better understanding of the invention, a description of the M protein structure is useful. See, Scientific American, June 1991, Streptococcal M Protein by Vincent A. Fischetti. Considering a typical M protein structure such as that of type M6, approximately 80 percent of the M6 molecule is made of four distinct regions, each of which consists of repeated sequences of amino acids. These regions are arbitrarily designated by the letters A through D. Near the N-terminal, or amino end, the part of the molecule farthest from the bacterial cell, lies region A. This region has five tandem repeats, or blocks, of 14 amino acids each. The three central repeats are identical, whereas the repeats at each end of the region diverge slightly from the common amino acid sequence. Next on the molecule is region B, which has a similar five-repeat structure except that the repeated blocks contain 25 amino acids. Region C consists of two and a half tandem repeats of 42 amino acids each; these blocks are not as identical to one another as those in the A and B repeats. Region D is composed of four partial repeats containing seven amino acids. The section buried in the cell extends from about the last repeat of the C region to the C-terminus.
Adjacent to the D-repeat blocks is a non-repeat region containing an abundance of proline and glycine amino acids, which are distributed in a nearly regular pattern. Beyond that region lies the C-terminal, or carboxyl, end of the molecule, which is the part within the cell. Near the C-terminal end are 20 hydrophobic amino acids and, at the terminus, six charged amino acids.
Similar arrangements of repeat blocks occur in the M proteins from type 5, 12, 24 and other streptococci. An alignment of the amino acid sequences of these different M proteins reveals that their C-terminal ends are more than 98 percent identical. Closer to the N-terminus, however, differences in sequence among M proteins increase. Consequently, the A-repeat blocks and a short amino acid region of about 10 to about 20 amino acids at the N-terminus are unique for each M molecule. This uniqueness is the major determinant of the sero-specificity of the immunological response.
In the amino acid sequence of M6 and later discovered in other M protein, another intriguing structural detail revealed itself. Running throughout all the repeat regions is an unusual seven-amino acid pattern: the amino acids in the first and fourth positions are hydrophobic; the intervening amino acids allow the protein to twist itself into a spiral shape called an alpha helix.
The seven-unit pattern in the arrangement of the amino acids in M6 indicates that the repeat regions of the protein molecule make up a long helical rod. The pattern in M6 is not perfect, nor is that pattern found in many other coiled-coil structures. Such irregularities probably account for the flexibility of the M molecules observed in electron micrographs. More important, the characteristics of these irregularities differ in the A-, B- and C-repeat regions. This observation suggests that each repeat region evolved independently and may have a distinct function. For an illustration of the protein sequence of M6 determined by cloning its gene, and for different forms of related M proteins when mutant streptococci delete copies of the amino acid repeats found in the parental molecule, especially in the N-terminus, see Scientific American, cited above. Studies have shown that each M protein fiber on a streptococcal cell wall is about 50 to 60 billionths of a meter long and consists of a single coiled-coil dimer (two M proteins coiled around each other).
It is likely that M proteins of all serotypes are built along a basic theme; they have a lengthy coiled-coil rod region in their centers that is flanked by a floppy section at the N-terminal end and an anchoring region at the C-terminal end. Because the alpha-helical coiled-coil structure can accommodate a large number of varying amino acid sequences, many different M proteins with the same general conformation can be constructed, as is shown hereinafter.
For an M protein to protect a streptococcus, it must be able to attach to the organism. The mechanism that holds surface proteins on gram-positive bacteria is still poorly understood, but various studies of the M protein have been enlightening in that respect.
It is believed that the 20-hydrophobic amino acids near the C-terminal end are positioned into the similarly hydrophobic membrane itself, whereas the charged amino acids at the very terminus protruded into the aqueous cytoplasm. Because the charged amino acids would resist moving into a hydrophobic environment, they would act like a knot at the end of a string, preventing the M molecule from being pulled through the membrane. That mechanism may be valuable for some proteins attached to membranes. More recent evidence indicates, however, that the attachment mechanism for M protein and other bacterial surface proteins may actually be more sophisticated. Studies have revealed that all surface proteins from gram-positive bacteria have a similar arrangement of hydrophobic and charged amino acids at their C-terminal end. See for instance Fischetti et al., Surface Proteins from Gram-Positive Cocci Share Unique Structural Features, New Perspectives on Streptococci and Streptococcal Infections, (G. Orefici, Editor), Gustav and Jena (Publishers) 1992.
More important, however, a short six-amino acid sequence adjacent to the hydrophobic region is highly conserved in all the known surface proteins of gram-positive bacteria. The sequence consists of a leucine, a proline, a serine, a threonine, a glycine and a glutamic acid. Its designation is usually abbreviated as LPSTGE (SEQ ID NO:23).
The importance of the LPSTGE (SEQ ID NO:23) sequence in the attachment of the M protein (and probably in all other proteins with this sequence motif) was shown by reported genetic experiments. It was found that if only the LPSTGE (SEQ ID NO:23) sequence is removed from the M protein gene, the M molecule that was produced would not attach to the bacterial membrane. This result suggested that the hydrophobic domain and the charged amino acids at the C-terminus are not sufficient for membrane attachment and that the LPSTGE motif may be an important signal for initiating the process.
In nearly all surface proteins found in gram-positive bacteria, there is another distinctive region that spans about 50 to 75 amino acids on the N-terminal side of the hydrophobic region. This part is probably located within the peptidoglycan. Proline, glycine, threonine and serine constitute a high percentage of these amino acids. The reason-for their prevalence has not been fully explored, but it is thought that prolines and glycines can create turns and bends in proteins. One hypothesis holds that cross-links in the peptidoglycan can weave through the proline- and glycine-induced bends, thereby stabilizing the M protein's position in the cell wall.
The knowledge that all known surface proteins on gram-positive bacteria attach themselves by a similar mechanism may open new avenues, such as controlling infections caused by these organisms. Surface proteins help pathogenic organisms initiate infections. It has been proposed that by preventing the proteins from anchoring to the bacterial cell, one should eventually be able to block infections and circumvent some of the problems associated with resistance to antibiotic therapies.
Just as the structures at the C-terminal end of the molecule provide information on how the M protein attaches to the bacterial cell, structures at the N-terminal end offer clues about how the molecule helps to fend off phagocytes. The N-terminal end of all M molecules has an excess of negatively charged amino acids, which results in a net negative charge for the region. Mammalian cells also exhibit a net negative charge on their surface. It has been suggested that the charge on M proteins may thus have evolved to hamper contact between streptococci and phagocytic cells through electrostatic repulsion. It has been proposed that one function of the central rod in the M protein is to act as a shaft for holding the negatively charged N-terminal end--and phagocytes--away from the bacterial surface.
At the N-terminal end of the coiled-coil rod, there is also a hypervariable region. This part of the molecule has a distinctive sequence in each M serotype. The hypervariable region consists of the short 10-30-amino acid non-helical sequence and if present, the adjoining A-repeat region. The hypervariable region plays an important role in the biological activity of the molecule; antibodies against this area are optimal at promoting phagocytosis and killing of the streptococci. This observation again explains why only serotype-specific antibodies protect against strep infections.
One hallmark of rheumatic fever is the presence of antibodies that react with muscle tissue, particularly heart tissue, in a patient's serum. See "Rheumatic Fever" by Earl H. Freimer and Maclyn McCarty; Scientific American, December 1965. Normally, antibodies are not made against one's own tissues. Researchers have discovered, however, that so-called cross-reacting antibodies can sometimes be induced by a molecule in an infective organism that resembles one in the mammalian host. In the process of making antibodies against the microbial molecules to clear an infection, the body is tricked into generating antibodies against its own tissues (serological cross-reactivity), a potentially harmful development.
It is evident from this description that there is an important and urgent need for a vaccine which is effective against the various serotypes of group A streptococci. The vaccine should be capable of raising sero-specific antibodies, especially those capable of triggering acute rheumatic fever, without eliciting cross-reaction with human tissue. There is also an important need for a vaccine which has not only these properties but also is capable of raising protective antibodies against infections, sore throat, skin infections, deep tissue infections and the like that are not necessarily but frequently are followed by rheumatic fever. The invention contributes to solving these important needs in human health.
Thus, there is an important need for a vaccine effective against streptococci infections which provides humoral immune against the diverse serotypes of group A streptococci and, when desired, also cellular immune responses. The vaccine should not elicit antibodies which react with human heart tissue.
In conjunction with studies of the M protein of various serotypes, it has been found that in most cases the protective epitopes of M protein may be separated from the potentially harmful, autoimmune epitopes of the molecule (see Refs. 5-7). The NH.sub.2 -terminal segments of M proteins have been found to evoke antibodies with the greatest bactericidal activity.
Further studies have shown that synthetic peptides copying limited regions of types 5, 6 and 24 M proteins evoked type-specific, opsonic antibodies that were not heart tissue cross-reactive. Because of their lack of immunogenicity, however, it was necessary to chemically link the synthetic peptides covalently to carrier proteins (see Refs. 5-7). Such fragments of M proteins linked to carrier proteins with chemical reagents do not result in hybrid proteins of defined structures. Thus, it has not been possible to obtain antigens which can elicit specific, desired antibodies without causing an increase of the risk of undesirable side reactions. Further, formation of hapten--carrier complexes using chemical cross-linking reagents is time-consuming and costly and results in undefined heterogenous mixtures of vaccine components. This invention provides multivalent vaccines that are type-specific and do not have the drawbacks of the prior art.